This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. CTA SAX-HPLC The samples were dissolved in water (20 mg/mL) and 10 [unreadable]L was injected onto a Phenomenex Gemini-NX C18 column (3 [unreadable]m, 250x4.6 mm), coated with cetyltrimethylammonium hydrogen sulfate. Analytes were detected by their UV absorbance at 232 nm. Separation was effected by a salt gradient (Table 1) using the following system: Solvent A: Water (brought to pH 3.0 by addition of conc. methylsulfonic acid);Solvent B: 2.5 M methylsulfonic acid, pH 2.5. Preparative Gel Filtration Twenty-five [unreadable]L of a 100 mg/mL aqueous solution of LMWH was injected onto two TSKGel G2000SW columns (7.8 mm ID X 30 cm, 5 [unreadable]m), connected in series and equipped with a TSKGel guard column (6.0 mm ID X 4.0 cm, 7 [unreadable]m), using 0.25 M ammonium acetate, pH 6.0 as eluent at a flow rate of 0.5 mL/min. Detection was by UV at 232 nm.. The fractions containing tetrasaccharide (41.8-42.9 min) were freeze-dried 3 times from water, redissolved in 25 [unreadable]L water, and repurified on the same column. The tetrasaccharide containing fractions were freeze-dried 3 times from water. ESI Mass Spectrometry Mass spectrometry was performed on a Thermo Scientific LTQ Orbitrap Discovery instrument in ESI mode under direct infusion. Infusion rate was 5 [unreadable]L/min, capillary temperature was 300 [unreadable]C, capillary voltage -13 V, spray voltage -3.2 kV, and sheath gas flow rate was 18. Both the ion trap and the Orbitrap FT detector were used to acquire mass spectra.